Summary: Facial clefts are one of the most common birth defects, affecting 2 out of every thousand babies. While the main causes of facial clefts are unknown, it is obvious that genetics plays a strong role. We've shown that families with one member affected by clefts are 40 times as likely to have a baby with a cleft. Environmental factors are also presumed to play a role in clefts. For example, clefts are easily produced in experimental animals exposed to teratogens. It is likely that humans vary in their genetic susceptibility to teratogens that cause clefts. More than a decade ago, we anticipated that genetic susceptibility would become an important area of epidemiologic research at NIEHS. Accordingly, we selected facial clefts as a condition with both genetic and environmental causes, and we began in 1992 to develop a study to address the causes of clefts. In 1996 we launched a population-based case-control study of facial clefts in Norway. (Norway has one of the highest rates of cleft lip and palate in the world.) The field phase was completed in 2002. We enrolled 88% of all babies with facial clefts born in Norway between 1996 and 2002 (574 cases), and 76% of eligible control infants (763) selected randomly from the population. Mothers of these infants provided detailed information on occupational and other exposures during pregnancy, as well as on nutrition, personal habits and medical history. Biological samples for DNA analysis were collected from cases and controls as well as their mothers, fathers and siblings. These total nearly 4000 people. DNA has been extracted from these samples and is now ready for genetic analysis. In the course of carrying out this study, we developed and published a new statistical strategy for the analysis of genetic data in case-parent triads that has been widely adapted. We have demonstrated the application of this new method in a preliminary analysis of 262 case-parent triads. We are working intensively on analyses of folic acid, cigarette smoking and hazardous occupations, and their effects on the risk of facial clefts. Parents? occupational exposures We have information on mother?s and father?s employment during the first trimester of pregnancy. Previous studies have identified occupational risks for clefts among service-related industries, agriculture and manufacturing. We coded employment using standard Nordic occupation and industry classifications. In an analysis led by Ruby Nguyen, my post-doctoral fellow, employed women who worked in manufacturing were found to have a slightly higher risk than other employed women (OR=1.3, 0.73-2.3). Adjustment for smoking, folate intake, income, year, and maternal education reduced this risk slightly (aOR=1.2, 0.65-2.3). This excess risk was present only among babies with cleft palate only (CPO) (aOR=1.9, 0.82-4.2). When we restricted the sample to women working full time, the aOR for CPO increased somewhat (aOR=2.7, 0.95-7.8). After excluding cleft palate cases that also had non-facial birth defects, the adjusted risk was 4.7 (1.6-7.3). With only 51 exposed women, we were unable to consider specific chemical exposures. If this association is not due to chance, it may reflect chemical exposures or perhaps unmeasured effects of shift-work or noise. A previous European study had reported increased risk with mother?s employment in manufacturing, but only for CLP and not CPO. Previous studies had identified beauty care workers as having increased risk for CL+P. There was an increased risk in our data as well, although the number of exposed mothers was small and the estimate imprecise (aOR=1.9; 0.57-6.2). After excluding cases with other defects, the aOR was 6.2 (1.0-38). We also asked women about their husbands? occupation and industry. While we don?t expect substantial associations with father?s exposures, this information has been reported in previous studies and will be incorporated into our analysis. Folic acid and clefts Low maternal folate causes neural tube defects, and folate supplementation reduces the risk. Researchers have proposed that folate might also help to prevent facial clefts. This hypothesis is not without some biological plausibility (the mid-facial structures of the lip and palate are formed from the same embryonic neural crest cells that create the neural tube), but epidemiologic data have been inconclusive. With this hypothesis in mind, we designed our case-control study to collect detailed folate exposure data during pregnancy. There are two main sources of folate: vitamin supplements and diet. We asked women about their use of folate supplements before and during pregnancy. Women who took supplements were asked to send an empty bottle so that we could confirm the dose. We also asked for bottles from other vitamin supplements, some of which contained unreported folate. To be consistent with earlier studies, we chose for our analysis to define the exposure window as the month before pregnancy and the first two months of pregnancy. We used the food frequency questionnaire to estimate dietary sources of folate in the first trimester. Dose categories for folate supplementation were chosen a priori on the basis of the frequency distribution of dose, with modes at 100 ug and 400 ug per day. Half of control women took no supplement, about one-quarter were in the low dose group, and one-quarter were in the high dose group. We see strong effects of folate on the risk of cleft lip with or without cleft palate (CL+P). We adjusted these risks by smoking, dietary folate, income, mother?s education, and year of case birth. Adjustment weakened the association with CPO, but had virtually no effect on the CL+P associations. Women who took 250 ug or more had an aOR of 0.60 for CL+P (95% CI = 0.42 ? 0.85) compared with all others. Excluding clefts with other defects did not change the results. Other vitamin supplements were unrelated to either type of cleft. Dietary folate had similar effects. The figure shows crude (unadjusted) risks. There was a consistent dose-response pattern across quartiles for cleft lip and palate, and a weaker pattern for cleft palate only. Once again, adjustment had little influence. Adjusting for the same variables as above and adding folate supplement to the model, women in the highest quartile had a risk of 0.67 for CL+P (0.47 ? 0.97) compared with the lowest quartile. The risk for CPO among women in the highest dietary quartile was 0.74 (0.48 ? 1.14). Once again, excluding clefts cases with other defects did not change the results. The two sources of folate were nearly independent in their effects. When we combine the two sources, the risk of CL+P was 0.43 (0.27 ? 0.69) among folate users (250+ mg) who were above the median in dietary folate, compared with women who took less than 250 mg folate and were below the dietary median. For cleft palate only, this risk was 0.56 (0.33 ? 0.96). These results are robust to minor changes in the exposure window. These data provide the strongest evidence to date that folate deficiency is a cause of facial clefts. We plan to pursue this finding in more detail through the assay of folate metabolism genes, which will allow us to look within genetically susceptible groups.